Method, reagent and kit for the detection of fecal occult blood

ABSTRACT

A fecal specimen may be tested for the presence of occult blood by combining the fecal specimen with 
     (a) an oxidizable substrate that produces a colored product in the presence of peroxide and hemoglobin; 
     (b) hydrogen peroxide or a peroxide source; and 
     (c) an enhancer selected to enhance the sensitivity of the test. Suitable enhancers are monocyclic nitrogen-containing aromatic heterocyclic compounds; tertiary or quaternary ammonium compounds having a phenyl, hydroxy alkyl or esterified hydroxy alkyl attached to the nitrogen; or quinoline or a substituted derivative thereof. If hemoglobin is present in the fecal sample, the oxidizable substrate is converted to the visibly detectable, colored product.

SPECIFICATION

This application is a divisional of U.S. patent application Ser. No.08/121,072 filed Sep. 14, 1993, now U.S. Pat. No. 5,447,868 issued Sep.5, 1995.

This invention relates to the detection of fecal occult blood, and inparticular to the use of enhanced developers in the detection of fecaloccult blood.

Fecal occult blood may provide a reliable diagnostic indicator of avariety of medical conditions involving gastrointestinal bleeding whichmay otherwise be difficult to detect, including colorectal cancer. Theuse of this method is well described in the medical literature. Seee.g., Greegor, D. H., Cancer 19:330-337 (1969) and Hastings, J. B.,Amer. J. Surg. 127:228-233 (1974). Tests for fecal occult blood basedupon the oxidation of guaiac to form a blue colored product in thepresence of hydrogen peroxide and hemoglobin have been described in U.S.Pat. No. 3,996,006, incorporated herein by reference. Such products aresold under the trademarks HEMOCCULT® AND SERACULT®. Briefly, the testinvolves placing a fecal sample on an absorbent paper coated with guaiacand adding a developer solution containing hydrogen peroxide. Ifhemoglobin is present, the guaiac is oxidized, turning the paper blue.

It is considered desirable to increase the sensitivity of the test inorder to detect excessive blood loss into the bowel at the earliestpossible stage. The occurrence of false negatives, the problem of notdetecting the presence of blood, is thus detrimental. Attempts havetherefore been made to increase the sensitivity of the test. Forexample, the addition of a drop or two of water to the fecal smear onthe guaiac impregnated test paper prior to the addition of the peroxidesolution has been used to enhance the test sensitivity. Such proceduresfrequently give a positive reaction even if there is no pathologicallysignificant amount of blood present in the stool. The occurrence offalse positives is also undesirable, since it can lead to unnecessaryfollow-up medical procedures.

An improved version of this test is described in European PatentPublication No. 0 308 227. Phenols are added to the developer solutionto achieve an enhancement of the amount of colored product produced,resulting in a more sensitive assay.

SUMMARY OF THE INVENTION

It has now been found that compounds other than phenols, specificallytertiary and quaternary nitrogen compounds, can be used to enhance thesensitivity of a test for fecal occult blood. It is therefore an objectof the present invention to provide an enhanced method for testing offecal occult blood that makes use of (1) monocyclic nitrogen-containingaromatic heterocyclic compounds such as triazole, pyridine, pyrazine orsubstituted derivatives thereof; (2) tertiary or quaternary ammoniumcompounds containing at least one hydroxy alkyl or esterified hydroxyalkyl group or a phenyl or substituted phenyl group to the nitrogen or(3) quinoline or a substituted derivative thereof to enhance thesensitivity of the test.

It is a further object of the invention to provide a developer solutioncontaining an enhancer in accordance with the invention together withperoxide in an ethanol water carrier for use in testing for fecal occultblood.

It is still a further object of the invention to provide a kit fortesting for fecal occult blood. Such a kit includes an enhanceddeveloper composition containing an enhancer according to the inventionand an oxidizable substrate such as guaiac.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, a fecal specimen may be testedfor the presence of occult blood by combining the fecal specimen with

(a) an oxidizable substrate that produces a colored product in thepresence of peroxide and hemoglobin;

(b) hydrogen peroxide or a peroxide source; and

(c) an enhancer selected from the group consisting of monocyclicnitrogen-containing aromatic heterocyclic compounds or tertiary orquaternary ammonium compounds which enhance the sensitivity of the test.If hemoglobin is present in the fecal sample, the oxidizable substrateis converted to the visibly detectable, colored product.

A preferred oxidizable substrate for use in accordance with theinvention is guaiac which is the substrate used in commerciallyavailable test kits. Other substrates which are oxidized by peroxide inthe presence of hemoglobin to produce a colored product that can bevisually detected in the presence of fecal material may also be used.Suitable alternative materials include 3,3", 5,5-tetramethylbenzidine(see U.S. Pat. No. 4,562,043 incorporated herein by reference),guaiaconic acid A and other leuco dyes that are oxidized in the presenceof peroxide and hemoglobin (see U.S. Pat. Nos. 4,219,336 and 4,971,914incorporated herein by reference).

A simple and convenient way to conduct the test is to place theoxidizable substrate on a solid support. A preferred solid support is anabsorptive paper which has been impregnated with the oxidizablesubstrate. The oxidizable substrate needs to be present in amountssufficient to generate a detectable amount of colored product, generallyabove 0.5 gm/1000 in². In the coated paper used in commercial kitstoday, this amounts to about 0.75 gm/1000 in². Using a micropipet, asmall (e.g. 0.03 ml) aliquot of the fecal specimen is placed on thesupport. One or two drops of developer solution are then added, and theextent of color development, if any, is observed.

This developer solution contains hydrogen peroxide or a source ofperoxide such as cumene hydroperoxide and the enhancer in anethanol/water carrier. The developer solutions of the inventiongenerally contain 4 to 6% by weight of hydrogen peroxide, 0.2 to 4% byweight of the enhancer, and 80 to 90% ethanol with the balance beingwater. Preferably, the enhancer will be present in amounts of from 0.3to 1.5% by weight, the exact amount depending on the specific enhancerbeing used. Unless limited by solubility considerations, higher enhancerconcentrations can also be used but it will be appreciated that theresulting increase in sensitivity may lead to the detection of lowlevels of occult blood that are not medically significant, i.e., falsepositives.

Enhancers useful in the present invention include monocyclicnitrogen-containing aromatic heterocyclic compounds such as triazole,pyridine, pyrazine and substituted derivatives thereof, including4-benzyl pyridine, 2-methoxy pyridine, 4-(p-nitrobenzyl) pyridine, andpyrazine carboxylic acid. Other enhancers in accordance with theinvention are tertiary or quaternary amines having at least one hydroxyalkyl or esterified hydroxy alkyl group attached to the nitrogen.Examples of such enhancers include diethyl ethanolamine, ethyldiethanolamine and triethanolamine, acetylcholine chloride and β-methylacetylcholine chloride. A further class of enhancers in accordance withthe present invention are tertiary and quaternary amines having a phenylgroup attached to the nitrogen, e.g. dimethyl aniline. Quinoline andderivatives thereof such as alpha-hydroxy quinoline were also found tobe effective enhancers.

EXAMPLE 1

Various compounds were tested for their ability to act as enhancers ofguaiac oxidation by peroxide in the presence of hemoglobin. A sample ofblood was diluted 1:20,000 to approximate the level of blood in a fecalsample and an 0.03 ml drop was placed onto a guaiac coated paper supportof the type used in SERACULT® and SERACULT PLUS® test kits. 1-2 drops ofa developer solution (0.04 to 0.1 ml) containing 4% H₂ O₂, 4% of aselected enhancer, 8% water and 84% ethanol was then placed on the papersupport and observations for color formation were made. Each enhancerwas rated on a scale of 1 to 4 for color formation, with 1 being theamount of color generated using no enhancer and 2 being the amount ofcolor generated using 1.5% ethyl hydroxybenzoic acid (ethyl paraben), aphenolic enhancer of the type disclosed in EP 0 308 227 and used incommercial products. The results of these tests are shown in Table 1. InTable 1, an observable color intensity less than that of the unenhancedcontrol is reported as TR.

During these tests, it was observed that the colored product producedvaried in stability depending on the enhancer. The rate of fading isalso reported in Table 1(VS=very slow, S=slow, M=moderate, F=fast). Ingeneral, a fade rate of moderate or slower is consistent with theordinary period for observing the results of fecal occult blood tests of30-120 seconds after testing.

                  TABLE 1                                                         ______________________________________                                                            Color                                                     Test Enhancer       Intensity                                                                              FADE                                             ______________________________________                                        2 - methoxy pyridine                                                                              3.5      S                                                3 - hydroxypyridine 2.5      S                                                3 - hydroxypyridine-N-oxide                                                                       1        S                                                4 - benzyl pyridine 4        S                                                pyridine            3.5      S                                                pyrazine            4        S                                                1,2,4 - triazole    3.5      S                                                pyrazine carboxylic acid                                                                          4        S                                                quinoline           3.5      S                                                2 - amino pyridine  tr       F                                                4 - (p-nitrobenzyl)-pyridine                                                                      3.5      S                                                (light yellow)                                                                pyridine - HCl      2.5      F                                                dimethyl ethanolamine                                                                             3.5      S                                                triethanolamine      1+      M-S                                              phenyl diethanolamine                                                                             3.5      S                                                8-hydroxy quinoline 3        S                                                Comparative Compounds                                                         phenyl salicylate   4        S                                                salicylic acid      tr       S                                                hydroquinone        0                                                         methylether hydroquinone                                                                          tr       S                                                dimethyl hydroquinone                                                                             1        S                                                p-phenyl phenol     2.5      VS                                               1,2 - dimethoxyphenol                                                                             tr       S                                                salicylamide        1.5      S                                                ______________________________________                                    

EXAMPLE 2

An 0.03 ml drop of 1:30,000 diluted blood sample was placed on guaiaccoated paper. One to two drops of a developer solution containing 84%ethanol, 4% hydrogen peroxide and 0.7% pyrazine carboxylic acid, thebalance being water, was placed on the test paper. A blue coloration wasobserved where the blood sample had been placed on the test paper.

EXAMPLE 3

An 0.03 ml drop of a 1:30,000 dilution blood sample was placed on aguaiac-coated test paper. One to two drops of a developer solutioncontaining 84% ethanol, 4% hydrogen peroxide, 0.5% 4-benzyl pyridine,the balance being water was then placed on the paper. A blue colorationwas observed where the blood sample had been placed on the test paper.

EXAMPLE 4

Additional enhancers were tested using a 1:25,000 dilution of a bloodsample. The results are summarized in Table 2.

                  TABLE 2                                                         ______________________________________                                                        Color                                                         Enhancer        Developed Comments                                            ______________________________________                                        1% acridine     2         developer is yellow                                                           but does not disturb                                                          result                                              1% dimethylethanolamine                                                                       2.5                                                           1.3%            2.5                                                           diethylethanolamine                                                           4% diethylethanolamine                                                                        1.5       faded fast                                          1% phenyldiethanolamine                                                                       4                                                             1% dimethyl aniline                                                                           2.5       faded fast                                          4% triethanolamine                                                                            1                                                             4.3% acetyl choline                                                                           1.5                                                           chloride                                                                      4% beta-methyl acetyl                                                                         1.5                                                           choline chloride                                                              4% acetyl choline                                                                             .5                                                            bromide                                                                       Controls                                                                      Seracult        <.5                                                           Seracult + 1.5% ethyl                                                                         1                                                             paraben                                                                       ______________________________________                                    

EXAMPLE 5

Because peroxide may be lost from developer solutions on aging,developer solutions with lower levels of peroxide were tested. Threesets of developer solutions were prepared for each enhancer testedcontaining 2%, 3% and 4% hydrogen peroxide by mixing 44 grams ethanol,0.04 g phosphoric acid, 0.3 to 2 grams of an enhancer and 0.32, 0.47. or0.63 grams of peroxide and adding water to a total weight of 50. Theresults, summarized in Table 3, show that lower peroxide concentrationscan be effectively used.

                  TABLE 3                                                         ______________________________________                                                  Peroxide                                                                      Concentration  Persistence of                                       Enhancer    4%       3%      2%    Blue color                                 ______________________________________                                        ethyl paraben                                                                             3+       3+      3+    slow fade                                  (1.5%)                                                                        benzophenone (4%)                                                                         1+       1+      1+    slow fade                                  dimethyl    3+       3+      3+    fast fade                                  ethanolamine (1%)                                                             benzyl pyridine                                                                           4+       4+      4+    medium fade                                (1%)                                                                          pyrazine (1%)                                                                             4+       4+      4+    medium fade                                pyrazine      3.5+     3.5+    3.5+                                                                              slow fade                                  carboxylic acid                                                               (0.6%)                                                                        ______________________________________                                    

EXAMPLE 6

To evaluate the storage stability of developer solutions according tothe invention, developers containing various enhancers was refluxed for48 or 65 hours and tested for the level of peroxide. The averagestarting peroxide level was 4.27% The samples which had been refluxedfor 65 hours were also tested for their usefulness in developing a fecaloccult blood test. The results, which are summarized in Table 4, showthe stability of various enhancers in accordance with the invention,although triazole had poor results in this rather extreme test.

                  TABLE 4                                                         ______________________________________                                                   H.sub.2 O.sub.2                                                                        H.sub.2 O.sub.2                                                                        COLOR   COLOR                                               (%)      (%)      SCORE   SCORE                                               after 48 after 65 W/O     W/                                       Enhancer   hours    hours    REFLUX  REFLUX                                   ______________________________________                                        1.23% 4-benzyl                                                                           3.48     3.25     4+      3+                                       pyridine   3.16     3.5                                                       1% 4(p-nitro-                                                                            4.57     4.14     4+      3+                                       benzyl) pyridine                                                                         4.65     nd                                                        dimethyl-  nd       1.62     3+      3+                                       ethanolamine                                                                             1.93     1.57                                                      1.1% 1,2,4 <.082             4+      0                                        triazole                                                                      benzophenone                                                                             1.74     nd       1+      1+                                                  1.8      nd                                                        ______________________________________                                    

EXAMPLE 7

The test of Example 6 was repeated but with a 72 hour reflux period anddifferent enhancers. The results are summarized in Table 5.

                  TABLE 5                                                         ______________________________________                                                                     COLOR   COLOR                                              Initial  H.sub.2 O.sub.2 (%)                                                                     SCORE   SCORE                                              Peroxide after 72  W/O     W/                                       Enhancer  (%)      hours     REFLUX  REFLUX                                   ______________________________________                                        0.6% pyrazine                                                                           4.27     4.14      3+      2+                                       carboxylic acid    4.27                                                       no enhancer                                                                             4.27     1.88      2+      1+                                                          2.1                                                        Seracult ® Plus                                                                     4.09     2.62      3+      2+                                                          2.45                                                       SANSA ®                                                                             4.11     2.97      2-3+    2+                                       (Lot 5017C)        2.95                                                       ______________________________________                                    

I claim:
 1. A method for detecting occult blood in a specimen comprising combining the specimen with(a) an oxidizable substrate that produces a colored product in the presence of peroxide and hemoglobin; and (b) a liquid developer comprising hydrogen peroxide or a peroxide source, a carrier consisting essentially of water and ethanol, and an enhancer component, said enhancer component consisting of a material selected from the group consisting of tertiary and quaternary amines having a hydroxy alkyl or esterified hydroxy alkyl group attached to the nitrogen, wherein the oxidizable substrate and liquid developer are combined with the specimen in amounts effective to produce a visually detectable color change if medically significant amounts of blood are present in the specimen.
 2. A method according to claim 1, wherein the enhancer component is present in the carrier in amounts of from 0.2% to 4% by weight.
 3. A method according to claim 1, wherein the enhancer component is present in the carrier in amounts of from 0.3% to 1.5% by weight.
 4. A method according to claim 1, wherein the enhancer component is phenyl diethanolamine.
 5. A method according to claim 1 wherein the enhancer component is dimethyl ethanolamine.
 6. A composition for use as a developer in a test for occult blood based upon the oxidation of a substrate to a colored product comprising(a) hydrogen peroxide or a peroxide source; (b) an amount of an enhancer component effective to enhance the amount of colored product produced; and (c) a carrier consisting essentially of water and ethanol, wherein the enhancer component consists of a material selected from the group consisting of tertiary and quaternary amines having a hydroxy alkyl or esterified hydroxy alkyl group attached to the nitrogen.
 7. A composition according to claim 6, wherein the enhancer component is present in amounts of from 0.2% to 4% by weight.
 8. A composition according to claim 7, wherein the peroxide is present in an amount of from 4% to 6% by weight.
 9. A composition according to claim 6, wherein the peroxide is present in an amount of from 4% to 6% by weight.
 10. A composition according to claim 6, wherein the enhancer component is phenyl diethanolamine.
 11. A composition according to claim 6, wherein the enhancer component is dimethyl ethanolamine.
 12. A kit for the detection of occult blood comprising, in packaged combination,(a) an oxidizable substrate which is converted to a colored product in the presence of peroxide and hemoglobin; and (b) a liquid developer comprising hydrogen peroxide or a peroxide source and an enhancer component in an amount effective to enhance the conversion of the oxidizable substrate to the colored product in a carrier consisting essentially of ethanol and water, wherein the enhancer component consists of a material selected from the group consisting of tertiary and quaternary amines having a hydroxy alkyl or esterified hydroxy alkyl group attached to the nitrogen.
 13. A kit according to claim 12, wherein the enhancer component is present in the developer in an amount of from 0.2% to 4% by weight.
 14. A kit according to claim 13, wherein the peroxide is present in the developer in an amount of from 4% to 6% by weight.
 15. A kit according to claim 12, wherein the peroxide is present in the developer in an amount of from 4% to 6% by weight.
 16. A kit according to claim 12, wherein the enhancer component is phenyl diethanolamine.
 17. A kit according to claim 12, wherein the enhancer component is dimethyl ethanolamine. 